Nobuaki Yanai

Identification of Bach2 as a B-cell-specific partner for small maf proteins that negatively regulate the immunoglobulin heavy chain gene 3' enhancer.

Maf family transcription factors are important regulators in various differentiation systems. Putative Maf recognition elements (MAREs) are found in the 3′ enhancer region of the immunoglobulin heavy chain (IgH) gene. These elements are bound in B‐cell extracts by a heterodimeric protein complex containing both Bach2 and a small Maf protein. Analysis of normal hematopoietic cells revealed that Bach2 is specifically expressed in B cells. Bach2 is abundantly expressed in the early stages of B‐cell differentiation and turned off in terminally differentiated cells. Bach2 acts together with MafK as a negative effector of the IgH 3′ enhancer and binds to the co‐repressor SMRT (silencing mediator of retinoid and thyroid receptor). Hence the Bach2–small‐Maf heterodimer may represent the first example of a B‐cell lineage, and of a developmental stage‐restricted negative effector of the MARE in the IgH 3′ enhancer region.

Hepatocyte cell lines established from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.

To establish cell lines exhibiting differentiation phenotypes, the immortalized cell lines were rapidly established from the primary culture of different tissues of transgenic mice harboring SV40 temperature-sensitive large T-antigen gene. The established cell lines grew at permissive temperature (33 degrees C), but not at nonpermissive temperature (39 degrees C). Several different cell types could be rapidly immortalized and cloned from the adult transgenic mice tissues. Among those cell lines, the established hepatocyte cell lines (TLR cell lines) exhibited liver-specific morphological and biochemical properties, but their properties were not coupled with the growth condition modified by temperature. The hepatocyte cell lines showed an inducibility of P450IA1 by 3-methylcholanthrene as observed in rat livers and this liver-specific function was stable even after 6 months of culture by continuous passages.

mRNA Expression and Transport Characterization of Conditionally Immortalized Rat Brain Capillary Endothelial Cell Lines; a New in vitro BBB Model for Drug Targeting

Abstract Brain capillary endothelial cell lines (TR-BBB) were established from a recently developed transgenic rat harboring temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat) and used to characterize the endothelial marker, transport activity, and mRNA expression of transporters and tight-junction strand proteins at the blood-brain barrier (BBB). These cell lines expressed active large T-antigen and grew well at 33°C with a doubling-time of about 22-31 hr, but did not grow at 39°C. TR-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. Although the γ-glutamyltranspeptidase activity in TR-BBBs was ~13% of that of the brain capillary fraction of a normal rat, it was localized in the apical side, suggesting that it reflects the functional polarity of the in vivo BBB. The mRNA of tight-junction strand proteins such as claudine-5, occludin, and junctional adhesion molecule are expressed in TR-BBB 13. Drug efflux transporter, P-glycoprotein, with a molecular weight of 170 kDa was expressed in all TR-BBBs and mdr 1a, mdr 1b, and mdr 2 mRNA were detected in TR-BBBs using RT-PCR. Moreover, mrp1 mRNA was expressed in all TR-BBBs. Influx transporter, GLUT-1, expressed at 55 kDa was revealed by Western blot analysis. It had 3-O-memyl-D-glucose (3-OMG) uptake activity which was concentration-dependent with a Michaelis-Menten constant of 9.86 ± 1.20 mM. The mRNA of large neutral amino acid transporter, which consists of LAT-1 and 4F2hc was expressed in TR-BBBs. In conclusion, the conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB) had endothelial makers, expressed mRNA for tight-junction strand proteins and the influx and efflux transporters and produced GLUT-1, which is capable of 3-OMG transport activity.

Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene

Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large T-antigen and grew well at 33°C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37°C but not at 39°C. However, growth was restored when the temperature of the culture was lowered to 33°C Although significant amounts of large T-antigen were shown to be present in the cell culture at 33°C, there was less of this complex at 37°C and 39°C. TM-BBBs expressed the typical endothelial marker, von Willberand factor and exhibited acetylated lowdensity lipoprotein uptake activity. The alkaline phosphatase and γ-glutamyltranspeptidase activity in TM-BBBs were −10% and 50% to 80% of brain capillary fraction of normal mice, respectively. D-Mannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59±1.16 mmol/1. Moreover, P-glycoprotein (P-gp) with a molecular weight of −170 kDa was expressed in all TM-BBBs. Both mdr 1a and mdr 1b mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT-PCR) analysis. [3H]-Cyclosporin A uptake by TM-BBB was significantly increased in the presence of 100 μmol/1 verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature.

Characterization of the Amino Acid Transport of New Immortalized Choroid Plexus Epithelial Cell Lines: A Novel In Vitro System for Investigating Transport Functions at the Blood-Cerebrospinal Fluid Barrier

Purpose. To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat).Methods. Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37°C during the first period of 3 days. Cells were subsequently cultured at 33°C to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup.Results. Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 1∼5) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na+, K+-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33°C with a doubling-time of 35∼40 hr. [3H]-L-Proline uptake by TR-CSFB cells took place in an Na+-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by α-methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [3H]-L-proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was fivefold greater than that following application to the basal side. In addition, both Na+-dependent and Na+-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells.Conclusions. Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo.

A mouse bone marrow stromal cell line, TBR‐B, shows inducible expression of smooth muscle‐specific genes

We established an in vitro culture system which mimicked the differentiation pathway of smooth muscle cell, using TBR‐B, a bone marrow stromal cell line derived from transgenic mice harboring temperature‐sensitive SV40 large T‐antigen gene. TBR‐B cells have the potential to express smooth muscle‐specific genes including h1‐calponin, h‐caldesmon, SM22α and α‐actin, only after cultured in the differentiation medium for 2 weeks. The differentiation state of TBR‐B was well controlled by using different culture medium. Using this cell line, we also found that ascorbic acid is a potent factor inducing the expression of h1‐calponin and α‐actin. TBR‐B cells will serve as a useful tool for elucidating the regulatory mechanisms of smooth muscle‐specific gene expression, and for identifying compounds that regulate the differentiation state of vascular smooth muscle cells.

A novel stromal cell-dependent hematopoietic cell line established from temperature-sensitive SV40 T-antigen transgenic mice.

A novel primitive hematopoietic cell line, THS119, was established from lineage marker negative (Lin-)/Sca-1+ cells from bone marrow of temperature-sensitive (ts) SV40 T-antigen transgenic mice after lengthy passaging by coculture with TBR59 bone marrow stromal cells. THS119 cells exhibited immature primitive hematopoietic cells such as forming cobblestones underneath the stromal cell layers. They retained properties of hematopoietic stem cells as shown by expression of c-Kit, Sca-1 and CD34low, but lacked hematopoietic lineage surface markers of differentiated hematopoietic cells (Gr-1, TER119, Mac-1, CD3, B220). RT-PCR analysis showed that THS119 cells exhibited multiple expression of both earlier developmental markers of myeloid, lymphoid and the hematopoietic cell specific transcription factors. THS119 cells showed temperature-dependent growth reflecting ts T-antigen, and their maintenance was TBR59 stromal cell-dependent. The requirement of stromal cells could not be replaced by cytokines, however, an IL-3 or IL-7 dependent cell line was generated after prolonged culture of THS119 cells on the stromal cells in the presence of these cytokines, and these cytokine-dependent cell lines exhibited phenotypes similar to the parental cells in their gene expression. SCF/c-Kit interaction is one factor required for their maintenance, but involvement of other factor(s) in the conditioned medium of TBR59 stromal cells was suggested. A novel immature hematopoietic cell line, THS119, may provide an appropriate experimental system to resolve how hematopoietic cells are kept in a primitive phase within a hematopoietic microenvironment.

Thrombin and TGF-β promote human leptomeningeal cell proliferation in vitro

Abstract Some disorders of the central nervous system, such as trauma, meningitis, or subarachnoid hemorrhage (SAH), result in inflammation and fibrosis of the arachnoid membranes followed by hydrocephalus. To clarify the role of growth factors in the pathophysiology of arachnoid fibrosis, we investigated the response of leptomeningeal (LM) cells to growth factors elevated in the cerebrospinal fluid (CSF) of patients with subarachnoidal inflammation. We examined the proliferative responses of LM cells to thrombin, transforming growth factor-β (TGF-β epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), platelet derived growth factor (PDGF), tumor necrosis factor-β (TNF-β and interleukin 1-β (IL1-β Thrombin, TGF-β, EGF, aFGF and PDGF promoted LM cell proliferation. TGF-β enhanced the proliferative effect of thrombin and EGF on LM cells. These findings suggest that thrombin and TGF-β which may be elevated in CSF following SAH, may cause subarachnoid fibrosis and subsequent hydrocephalus.

Development of the conditionally immortalized testicular Sertoli cell line TTE3 expressing Sertoli cell specific genes from mice transgenic for temperature sensitive simian virus 40 large T antigen gene.

PURPOSE We developed and characterized a conditionally immortalized testicular Sertoli cell line from transgenic mice bearing the temperature sensitive simian virus 40 large T antigen gene pSVtsA58. MATERIALS AND METHODS Established cells from 8-week-old male transgenic mice were cultured at a permissive (33C) or nonpermissive (39C) temperature on a collagen type I pre-coated culture vessel. The expression of Sertoli cell specific proteins was analyzed by reverse transcriptase-polymerase chain reaction, immunocytochemical testing and Western blot analysis. RESULTS The Sertoli cell line TTE3 grew at 33C but not at 39C. Large T antigen was expressed only in the nuclei at 33C, indicating that the temperature sensitive growth phenotype of the cells arose as a result of the function of temperature sensitive simian virus 40 large T antigen. The cells did not show any colony forming activity in soft agar or form tumors in subcutaneous tissue in nude mice, showing that TTE3 cells were not transformed. The cells expressed messenger RNAs encoding steel factor, inhibin-alpha, transferrin, follicle-stimulating hormone receptor and sulfated glycoprotein-2. Moreover, expression of vimentin and zonula occludens-1 was observed in the cytoplasm and on the boundaries of the cells, respectively. Interestingly expression levels of transferrin and zonula occludens-1 were significantly elevated at 39C. CONCLUSIONS TTE3 cells with these unique characteristics should serve as a useful model to study the regulation of Sertoli cell function.

Regulation of myeloid and lymphoid development of hematopoietic stem cells by bone marrow stromal cells.

Development of hematopoietic stem cells is regulated by stromal cells of the bone marrow. Many stromal cell lines have been established from temperature-sensitive SV40 large T-antigen gene transgenic mice and used to examine regulation of the purified stem cells. When the sorted stem cells were cocultured on the stromal cell layers, cobblestone formation was induced by the stromal cells. The cobblestones were formed by finite cell division (8 divisions on average) of sorted Lin- c-Kit+ Sca1+ stem cells committed to myeloid or lymphoid lineages. These stromal cell lines showed variable activities supporting the stem cell development. In one stromal cell line, TBR59, two waves of cobblestone formation committed to either myeloid lineage or lymphoid lineage were induced. TBR31-1, another bone marrow stromal cell line, induced only the cobblestone formation committed to lymphoid lineage. These results indicate that the bone marrow stromal cells selectively induce lineage-specific commitment of the stem cells. Both cobblestone formations require c-Kit function as well as adhesive interaction through VLA4 and VCAM1.